Human embryonic stem (hES) cells have received considerable attention with regards to their regenerative capacity. However, basic biological pathways including apoptosis in hES cells remain largely unexplored. We have started to investigate the apoptotic pathway in hES cells and have uncovered novel and fascinating mechanisms by which hES cells regulate cell death. We found that hES cells are highly sensitive to DNA damage, with all cells dying by 6 hours. A critical mediator of apoptosis in mammalian cells is Bax. In most cells, Bax is maintained in the cytosol in an inactive conformation and becomes activated only in response to apoptotic stimuli. Activated Bax then translocates to the mitochondria to induce cytochrome c release and caspase activation. Remarkably, we found that hES cells maintain Bax in its already active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. Our results show that after DNA damage, active Bax rapidly translocated from the Golgi to mitochondria by a p53-dependent mechanism. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development. In this proposal, we will investigate this novel and unexpected mechanism by which apoptosis is regulated in hES cells. We will focus specifically on examining how Bax is maintained in an active state (Aim 1), determine how it localizes to the Golgi (Aim 2) and identify the molecular events triggered by DNA damage to induce the rapid translocation of active Bax from the Golgi to the mitochondria (Aim 3) in hES cells. These studies will undoubtedly uncover critical aspects of apoptosis regulation in cells and reveal key features of stem cell biology that can have significant impact for regenerative medicine.